Cholesteryl Ester Transfer Protein (CETP) Inhibitor and Pharmaceutical Compositions Comprising Said Inhibitor for Use in the Treatment or Prevention of Cardiovascular Diseases

ABSTRACT

The present invention relates to a cholesteryl ester transfer protein (CETP) inhibitor for use in the treatment of subjects suffering from or having an increased risk for cardiovascular diseases, in particular hyperlipidemia or mixed dyslipidemia. A further aspect of the present invention relates to a pharmaceutical composition for use in the treatment of subjects suffering from or having an increased risk for cardiovascular diseases, wherein the composition comprises a therapeutically effective amount of said CETP inhibitor.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a cholesteryl ester transfer protein(CETP) inhibitor and a pharmaceutical preparation comprising saidCETP-inhibitor for use in the treatment of subjects suffering from orhaving an increased risk for cardiovascular diseases, in particularhyperlipidemia or mixed dyslipidemia.

BACKGROUND OF THE INVENTION

Prospective epidemiological studies have shown a strong associationbetween low density lipoprotein-cholesterol (LDL-C) levels andcardiovascular disease (CVD) risk (1). The subsequent application ofstatin therapy to decrease these atherogenic LDL-C levels has resultedin a marked reduction of CVD-related morbidity and mortality: every 1mmol/L decrease in LDL-C results in an estimated 22% reduction of CVDevents and a 10% reduction of all-cause mortality (2). Notwithstandingthese impressive benefits, a large residual disease burden persists thathas a large impact on both individual patients as well as on globalhealthcare costs (3). Novel therapeutics are required to reduce furtherthis residual CVD risk in patients.

One new approach which reduces LDL-C and elevates HDL-C levels is toinhibit Cholesterol Ester Transfer Protein (CETP). CETP is a plasmaprotein secreted primarily by liver and adipose tissue. CETP mediatesthe transfer of cholesteryl esters from HDL to apolipoprotein B (ApoB)-containing particles (mainly LDL and VLDL) in exchange fortriglycerides, thereby decreasing the cholesterol content in HDL infavor of that in (V)LDL. Hence, CETP inhibition has been hypothesized toretain cholesteryl esters in HDL-C and decrease the cholesterol contentof the atherogenic Apo B fraction.

Despite the evidence supporting the potential of CETP inhibition inreducing cardiovascular morbidity, clinical development of CETPinhibitors has not been straightforward. The first compound to progressto phase 3 clinical trials was torcetrapib which was dosed at 60 mg.Torcetrapib was shown to increase HDL-C by 72% and decrease LDL-C by25%, but it was subsequently withdrawn from development owing to safetyconcerns including an unexpected increase in cardiovascular events anddeath when in combination with atorvastatin, compared with atorvastatinalone (11).

Although the mechanism of those events is not fully understood, there isincreasing evidence that they might have been due to off-target effectsof torcetrapib such as increased blood pressure, changes in electrolytes(increases in sodium and bicarbonate and decreases in potassium) andincreases in aldosterone, consistent with mineralocorticoid activity(11,12,13,14,15). There is also some evidence from animal studies thattorcetrapib increases expression of endothelin-1, which has beenpostulated to be have contributed to the apparent (non-significant)increase in cancer deaths in the ILLUMINATE trial (16,17). Theseobservations could be related to the relatively high dose oftorcetrapib.

Subsequently, another CETP inhibitor, dalcetrapib, entered phase 2bclinical trials. Dalcetrapib was shown to be a weak inhibitor thatincreased HDL-C by 30-40% with minimal effects on LDL-C concentrationsbut did not appear to exhibit the off-target effects of torcetrapib(18,19,20). Recently, dalcetrapib development has also been terminatedon the grounds of futility in a Phase 3 study where the drug was dosedat 600 mg. Lack of efficacy was probably related to modest CETPinhibition (18).

Two more CETP inhibitors, anacetrapib and evacetrapib, are currently inphase 3 clinical trials. Data from phase 2 studies suggest that both areCETP inhibitors without mineralocorticoid activity. Anacetrapib 200 mgonce daily has been shown to increase HDL C by 97% and decrease LDL-C by36% in fasted healthy subjects (21) and 150 mg once daily anacetrapibhas been shown to increase HDL C by 139% and decrease LDL-C by 40% inpatients (22). Evacetrapib (500 mg once daily monotherapy in patients)has been shown to increase HDL-C by 129% and decrease LDL-C by 36% (23).

In the ongoing Phase 3 studies, once daily dose of 100 mg anacetratib isbeing clinically evaluated, whereas for evacetrapib a once daily dose of130 mg is being evaluated. Such relatively high amounts of activeingredients may lead to several problems.

Due to the fact that a relatively high amount of the above-mentionedCETP-inhibitors has to be administered, the solid oral dosage forms,such as tablets or capsules, will be relatively big. This causesproblems with swallowing of such tablets and capsules. Alternatively,one may choose to administer multiple smaller tablets or capsules;however this has a negative influence on patient compliance and costs.

A further disadvantage of the use of the present CETP-inhibitors is thatdue to the relatively high dosage which has to be used to obtainCETP-inhibition, more and stronger side effects may occur. This can havea negative influence on both the physical well-being of the patient aswell as on patient compliance. Moreover, due to a lower bioavailabilityof the known CETP-inhibitors, inter-subject pharmacokinetic variabilitymay occur. Furthermore, since a relatively high dose is needed for theknown CETP-inhibitors (such as anacetrapib) to be effective, it willtake several years to eliminate these CETP-inhibitors from the body(reference The American Journal of Cardiology available online 4 Oct.2013: Evaluation of Lipids, Drug Concentration, and Safety ParametersFollowing Cessation of Treatment With the Cholesteryl Ester TransferProtein Inhibitor Anacetrapib in Patients With or at High Risk forCoronary Heart Disease Antonio M. Gotto Jr. et al.).

Hence, a need remains for the provision of a potent and well toleratedCETP-inhibitor and a pharmaceutical composition thereof, which does notshow the above mentioned disadvantages.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to the compound

(hereinafter referred to as Compound A) or a pharmaceutically acceptablesalt thereof for use in the treatment of subjects suffering from orhaving an increased risk for cardiovascular diseases, wherein the doseof Compound A administered to said subjects ranges from 1 to 25 mg perday.

A second aspect of the present invention relates to a pharmaceuticalcomposition for use in the treatment of subjects suffering from orhaving an increased risk for cardiovascular diseases, wherein thecomposition comprises a therapeutically effective amount of

Compound A or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient. The dose of Compound A to beadministered to the subjects with the pharmaceutical compositionaccording to the present invention preferably ranges from about 1 to 25mg per day.

Clinical studies have shown that Compound A is a potent CETP-inhibitor.Compared to other known CETP-inhibitors, only a relatively low dose ofCompound A is needed to reach near complete CETP inhibition. Typically,repeated once daily dosages as low as 2.5 mg of Compound A have provento be already sufficient to reach near complete CETP-inhibition. Theseare considerably lower dosages than had to be used for otherCETP-inhibitors.

Moreover, clinical studies have also shown that Compound A is welltolerated and that it does not lead to serious side effects. Forinstance, there were no clinically significant effects observed on bloodpressure or heart rate, nor does Compound A appear to have an effect onserum electrolyte or aldosterone concentrations. Clinical studies alsoshowed that Compound A does not suffer from food effects and that at theclaimed dose it does not show prolonged residual effects on cessation ofdosing.

A third aspect of the present invention relates to a pharmaceuticalcomposition per se, which composition comprises 1 to 25 mg of Compound Aor a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

A fourth aspect of the present invention relates to a method forpreparing such a composition.

DEFINITIONS

The term ‘pharmaceutical composition’ as used herein has itsconventional meaning and refers to a composition which ispharmaceutically acceptable.

The term ‘pharmaceutically acceptable’ as used herein has itsconventional meaning and refers to compounds, material, compositionsand/or dosage forms, which are, within the scope of sound medicaljudgment suitable for contact with the tissues of mammals, especiallyhumans, without excessive toxicity, irritation, allergic response andother problem complications commensurate with a reasonable benefit/riskratio.

The term ‘therapeutically effective amount’ as used herein has itsconventional meaning and refers to an amount or concentration which iseffective in producing the desired effect in a mammal, e.g., inreducing, eliminating, treating, preventing or controlling the symptomsof a disease or condition affecting a mammal, in particular human.

The term ‘controlling’ is intended to refer to all processes whereinthere may be a slowing, interrupting, arresting or stopping of theprogression of the diseases and conditions affecting the mammal.However, ‘controlling’ does not necessarily indicate a total eliminationof all disease and condition symptoms, and is intended to includeprophylactic treatment.

The term ‘excipient’ as used herein has its conventional meaning andrefers to a pharmaceutically acceptable ingredient, which is commonlyused in the pharmaceutical technology for preparing a granulate, solidor liquid oral dosage formulation.

The term ‘salt’ as used herein has its conventional meaning and includesthe acid addition and base salts of Compound A.

The term ‘increased risk’ has its conventional meaning and refers to asituation in a subject, preferably human, where in individuals, eithermale or female, have an LDL-cholesterol level above 2.6 mmol/l, suchthat they are exposed at an increased risk of a cardiovascular event,compared to those with lower levels.

The term ‘treatment’ as used herein has its conventional meaning andrefers to curative, palliative and prophylactic treatment.

The term ‘cardiovascular disease’ has its conventional meaning andincludes arteriosclerosis, peripheral vascular disease, hyperlipidemia,mixed dyslipidemia betalipoproteinemia, hypoalphalipoproteinemia,hypercholesteremia, hypertriglyceridemia, familial-hypercholesteremia,angina, ischemia, cardiac ischemia, stroke, myocardial infarction,reperfusion injury, restenosis after angioplasty, hypertension, cerebralinfarction and cerebral stroke.

The term ‘unit dosage form’ has its conventional meaning and refers to adosage form which has the capacity of being administered to a subject,preferably a human, to be effective, and which can be readily handledand packaged, remaining as a physically and chemically stable unit dosecomprising the therapeutic agent, i.e. Compound A.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to the compound:

(hereinafter referred to as Compound A) or a pharmaceutically acceptablesalt thereof for use in the treatment of subjects, preferably humans,suffering from or having an increased risk for cardiovascular diseases,wherein the dose of Compound A administered to said subjects ranges fromabout 1 to 25 mg per day.

Compound A as such has already been described in the European patentapplication EP1730152, wherein it has been identified as aCETP-inhibitor among many other CETP-inhibitors. Surprisingly, it hasnow been found that Compound A has exceptionally good pharmacodynamicand pharmacokinetic properties compared to other CETP-inhibitorsmentioned in EP 1730152 or used clinically in particular, Compound A hasa surprisingly better bioavailability than other known CETP-inhibitors.It has also been found that Compound A may effectively be usedclinically in a relatively low dose of about 1 to 25 mg per day,preferably 1 up to and including 10 mg per day. Such doses arepreferably administered as a pharmaceutical composition comprisingCompound A and an excipient. The prior art does not disclose or suggestthat it is possible to use CETP-inhibitors effectively at such a lowdose. In this regard reference is made to anacetrapib and evacetrapib,which both required in a clinical setting once daily doses of more than100 mg.

Preferably, a dose of about 5 to up to and including 10 mg of Compound Aper day is used, alternatively a dose of about 5 mg of Compound A, adose of about 10 mg of Compound A or a dose of about 25 mg of CompoundA.

Clinical studies have shown that within the claimed dosage range ofabout 1 to 25 mg per day it is possible to achieve near completeCETP-inhibition, significant increase of HDL-cholesterol concentrationand a remarkable decrease of LDL-cholesterol levels in subjects whichhave been administered Compound A. The clinical studies have also shownthat these effects already occur after a single dose of Compound A.

However, it is preferred to administer to a subject in need of CompoundA for extended periods of time the once daily dose of about 1 to 25 mg,preferably a once daily dose of about 5 to 10 mg. Preferably, thesubjects in need of Compound A are administered a daily dose of about 1to 25 mg, preferably about 5 to 10 mg, for 1, 5, 10, 20, 40 52, 100 or200 weeks.

It is particularly preferred to administer a dose of 1 to 25 mg per dayto a subject in need thereof, i.e. a person suffering fromcardiovascular diseases or a person having an increased risk forcardiovascular diseases for at least one week, preferably at least threeweeks.

Clinical studies have also shown that at a relatively low dose of about1 to 25 mg of Compound A per day, preferably about 5 to 10 mg per day,no serious adverse effects appeared. For instance, there were noclinically significant effects observed on blood pressure or heart rate,nor does Compound A at the claimed dose appear to have off-targeteffects, such as on serum electrolyte or aldosterone concentrations. Ithas also been shown that with the claimed daily dose of Compound A doesnot suffer from food effects and that at the claimed dose it does notshow prolonged residual effects on cessation of dosing due to incompletedrug washout.

The dose of about 1 to 25 mg of Compound A per day, preferably the doseof about 5 to 10 mg, is particularly suitable for use in the treatmentof persons suffering from or having an increased risk for cardiovasculardiseases, such as arteriosclerosis, peripheral vascular disease,hyperlipidemia, mixed dyslipidemia, hyperbetalipoproteinemia,hypoalphalipoproteinemia, hypercholesteremia, hypertriglyceridemia,familial-hypercholesteremia, angina, ischemia, cardiac ischemia, stroke,myocardial infarction, reperfusion injury, restenosis after angioplasty,hypertension, cerebral infarction, cerebral stroke.

In view of the remarkable decrease of CETP activity, the remarkabledecrease of LDL-cholesterol plasma concentration and the significantincrease of HDL-cholesterol plasma concentration, the lack of sideeffects and food effects, it appears that a daily dose of about 1 to 25mg, preferably 1 to 10 mg of Compound A is particularly suitable for usein the treatment of patients suffering from or having an increased riskfor mixed dyslipedimia, hyperlipidemia, or in particular primaryhyperlipidemia.

Besides Compound A as such, pharmaceutically acceptable salts thereofmay also be used. Pharmaceutically acceptable salts of Compound Ainclude the acid addition and base salts thereof, such as preferably thecalcium, potassium or sodium salts. For a review on suitable salts,reference is made “Handbook of Pharmaceutical Salts: Properties,Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany,2002).

A pharmaceutically acceptable salt of Compound A may be readily preparedby mixing together solutions of Compound A and the desired acid or base,as appropriate. The salt may precipitate from solution and be collectedby filtration or may be recovered by evaporation of the solvent. Thedegree of ionisation in the salt may vary from completely ionised toalmost non-ionised.

The present invention also relates to pharmaceutically acceptablesolvates of Compound A and to pharmaceutical compositions comprisingsuch solvates for use in the treatment of subjects suffering from orhaving an increased risk for said cardiovascular diseases.

Also within the scope of the invention are so called ‘prodrugs’ ofCompound A. Thus certain derivates of Compound A, which may have littleor no pharmacological activity themselves, can when administered intothe body be converted into Compound A having the desired CETP-inhibitoryactivity. Such derivates are within the context of the present inventionreferred to as ‘prodrugs’. Prodrugs in accordance with the inventioncan, for example, be produced by replacing appropriate functionalitiespresent in Compound A with certain moieties known to those skilled inthe art as ‘pro-moieties’ as described in for example “Design ofProdrugs” by H. Bundgaard (Elsevier, 1985).

The claimed dose of Compound A is preferably administered orally tosubjects in need thereof. Preferably, Compound A is administered bymeans of a pharmaceutical composition. Oral administration may involveswallowing, so that the compound enters the gastrointestinal tract.Alternatively, buccal or sublingual administration may also be employedwherein Compound A enters the blood stream directly from the mouth.Pharmaceutical preparations, as described below, may be developed whichfacilitate the oral administration.

A second aspect of the present invention relates to a pharmaceuticalcomposition for use in the treatment of subjects suffering from orhaving an increased risk for cardiovascular diseases, wherein thecomposition comprises a therapeutically effective amount of Compound Aor a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient. Compound A and its pharmaceuticalsalts or prodrugs may be as described above.

The dose of Compound A to be administered to the subjects with thepharmaceutical composition according to the present invention preferablyranges from about 1 to 25 mg per day, more preferably from about 5 to 10mg per day. Alternatively, a dose of about 5 mg of Compound A, a dose ofabout 10 mg of Compound A or a dose of about 25 mg of Compound A isused.

As already described above, clinical studies have shown that with such arelatively low dose of Compound A, a remarkable decrease of CETPactivity, a remarkable decrease of LDL-cholesterol plasma concentrationand a significant increase of HDL-cholesterol plasma concentration isreached. Furthermore, it has also been shown that no serious adverseeffects occurred at such dose and that no food effects were observed andthat Compound A does not show prolonged residual effects on cessation ofdosing.

The pharmaceutical composition for use according to the presentinvention is preferably administered to the subject in need thereof for1, 5, 10, 20, 40, 52, 100 or 200 weeks. It is particularly preferred toadminister the pharmaceutical composition to a subject in need thereoffor at least one week, preferably at least three weeks.

In a preferred embodiment of the present invention, the pharmaceuticalcomposition is formulated as a single unit dosage form. The single unitdosage form is preferably a solid oral dosage form, such as a tablet orcapsule. Preferably, the single unit dosage form comprises about 1 to 25mg of Compound A, preferably about 5 to 10 mg of Compound A. It isparticularly preferred to use a solid oral dosage form such as tablet orcapsule comprising about 1 to 25 mg, preferably about 5 to 10 mg ofCompound A.

Solid oral dosage forms which may be used within the context of thepresent invention include besides tablets and capsules amongst otherscaplets, lozenges, pills, mini-tablets, pellets, beads and granulespackaged in sachets. Liquid oral dosage forms which may be used for thepharmaceutical preparation of the present invention include, but are notlimited to drinks, solutions, beverages and emulsions.

The pharmaceutical composition for use in the present inventioncomprises besides Compound A also an excipient, i.e. a pharmaceuticallyacceptable ingredient, which is commonly used in the pharmaceuticaltechnology for preparing granulate, solid or liquid oral dosageformulations.

Examples of categories of excipients include, but are not limited to,binders, disintegrants, lubricants, glidants, fillers and diluents. Oneof ordinary skill in the art may select one or more of theaforementioned excipients with respect to the particular desiredproperties of the granulate and/or solid oral dosage form by routineexperimentation and without any undue burden. The amount of eachexcipient used may vary within ranges conventional in the art. Thefollowing references which are all hereby incorporated by referencedisclose techniques and excipients used to formulate oral dosage forms.See “The Handbook of Pharmaceutical Excipients”, 4th edition, Rowe etal., Eds., American Pharmaceuticals Association (2003); and “Remington:The Science and Practice of Pharmacy”, 20th edition, Gennaro, Ed.,Lippincott Williams & Wilkins (2000).

A third aspect of the present invention relates to a pharmaceuticalcomposition per se comprising about 1 to 25 mg of Compound A or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier. Preferably, the pharmaceutical composition comprises5 to 10 mg of Compound A or a pharmaceutically acceptable salt thereof.

Compound A and its pharmaceutical acceptable salts and possible prodrugsmay be in the forms as described above.

The pharmaceutical composition is preferably formulated as a single unitdosage form as described above. More preferably, the composition isformulated as a liquid oral dosage form or as a solid oral dosage form,most preferably a tablet or capsule.

In a preferred embodiment the pharmaceutical composition comprises atablet or capsule comprising about 1 to 25 mg, preferably 5 to 10 mg ofCompound A or a pharmaceutically acceptable salt thereof.

A fourth aspect of the present invention relates to a method forpreparing the above mentioned pharmaceutical compositions.Pharmaceutical compositions of Compound A may be prepared by meanscommonly known to the person skilled in the art.

The present invention will be illustrated further by means of thefollowing non-limiting examples

EXAMPLES

In the following examples Compound A was studied in an in vitro assay,ex vivo and clinically. For synthesizing Compound A the method describedin the international patent application WO2005095409 was used.

Example 1: In Vitro and Ex Vivo Experimental Method of In Vitro Assay(a) Human Plasma Preparation

Human blood was obtained from healthy male volunteers using 0.1% EDTA asan anticoagulant, and centrifuged at 3,000 rpm for 15 minutes at 4° C.Human plasma was pooled, and then used for the preparation of ³H-labeledHDL or stored at −80° C. for CETP assay until use. The ³H-labeled HDLwas prepared with human plasma as described by Glenn and Melton (Methodsin enzymology. 263; 339-351, 1996). The specific gravity of plasma wasmeasured by a hydrometer, and the density was adjusted to 1.125 g/mL byadding solid KBr. After centrifugation at 100,000 rpm for 4 hours at 12°C. (rotor: 100.4, Optima TLX, Beckman), d>1.125 g/mL fraction wasdialyzed against 4 L of Tris-saline-EDTA buffer (TSE; 50 mmol/L Tris,150 mmol/L NaCl, 2 mmol/L EDTA, pH 7.4) for 18 hours at 4° C.[1,2-³H(N)]-Cholesterol (37 MBq/mL) was added to the dialyzed plasmafraction at the amount of 2 μCi/mL. The tube was tightly sealed under N₂gas stream and incubated at

37° C. for 18 hours with gently stirring to allow esterification ofradio-labeled cholesterol by endogenous LCAT. The incubated plasmafraction was adjusted to d=1.21 g/mL with solid KBr, and centrifuged at100,000 rpm, 12° C. for 5 hours. ³H-labeled HDL fraction was dialyzedagainst 2 L of TSE at 4° C. for 18 hours. The radioactivity of³H-labeled HDL was counted in a liquid scintillation counter. ³H-labeledHDL was stored at 4° C. until use.

(b) CETP Assay

CETP activity was determined as the rate of ³H-labeled CE transfer fromdonor HDL to acceptor VLDL/LDL. Human plasma (94 μL) was pre-incubatedwith compounds dissolved in DMSO (1 μL) 24 hours at 37° C., and thenincubated for 4 hours at 4° C. or 37° C. with 5 μL of ³H-labeled HDL.One hundred μL of phosphotungstate/MgCl₂ reagent (Wako pure chemical)were added to precipitate apoB-containing lipoproteins. Aftercentrifugation at 3,000 rpm for 10 minutes at room temperature, theradioactivity of the supernatant was counted in a liquid scintillationcounter. CETP activity was determined as the difference of theradioactivity between the samples incubated at 37° C. and 4° C. asfollows: % inhibition=100−{dpm (DMSO at 4° C.−test compounds at 37°C.)/dpm (DMSO at 4° C.−DMSO at 37° C.)}×100. The concentration achieving50% inhibition of CETP activity (IC₅₀) was estimated.

Experimental Method of Ex Vivo Assay (a) Compound Administration andCollection of Blood

Syrian Golden hamsters were used for the experiment after 1-weekacclimatization. After an overnight fast, animals were orally given thecompound suspension in 0.5% sodium carboxymethylcellulose in a volume of10 mL/kg. Under deep anesthesia with ether, blood was collected from theabdominal aorta 3 hours after the administration. For preparation ofserum, the collected blood was transferred to a plastic tube containinga clot activator, left still for 15 minutes at room temperature andcentrifuged. Serum CETP activities were determined immediately.

(b) Determination of Serum CETP Activity Ex Vivo

Ninety-five μL serum were added to 5 μL of 0.1 mM sodium phosphatebuffered saline (pH 7.0) containing 1.5 mM5,5′-dithio-bis(2-nitrobenzoic acid) in two 96-well V-bottom plates. Oneplate was incubated at 4° C. and the other was incubated at 37° C. After18 hours of incubation, each sample was mixed with 100 μL of reagent forprecipitation of apolipoprotein B-containing lipoproteins(phosphotungstate/MgCl₂ reagent, Wako pure chemical), left still for 10minutes at room temperature and centrifuged. Total cholesterol (TC) andfree cholesterol (FC) in supernatant were measured using commercial kits(Cholesterol E-test wako, and Free Cholesterol E-test wako; Wako purechemical). Cholesteryl ester (CE) was calculated by subtracting FC fromTC. CETP activity was determined by the following formula:

CETP activity=[CETP transfer]*/[CE value in 4° C. incubationsample]*CETP transfer=[CE value in 4° C. incubation sample]−[CE value in37° C. incubation sample]

(c) Results

in vitro IC₅₀ ex vivo (μM) (% human plasma inhibition) compoundStructural elements at indicated positions (24 h pre- 3 mg/kg example #R1 R2 R3 R4 incubation) (CMC) Compound A

CF₃ CH₃ CF₃ 0.064 79.2 1

CF₃ CH₃ CF₃ 0.21 57.5 2

CF₃ CH₃ CF₃ 0.23 38.4 3

CF₃ CH₃ CF₃ 0.45 5.4 4

CF₃ CH₃ CF₃ 0.79 8.6 5

CF₃ CH₃ CF₃ 1.7 6.2 6

CF₃ CH₃ CF₃ 0.22 13.9 7

CF₃

CF₃ 0.11 25.4 The core structure is:

Example 2: Double Blind Randomized Study of Subjects Receiving MultipleDoses of Compound A or Placebo Study Design

The clinical study was a repeated dose study in 5 groups of Caucasianmale subjects aged 18 to 55 years. Each subject received a single oraldose of Compound A/placebo on Day 1, followed by once daily doses onDays 8 to 35 (5 mg Compound A/placebo—Group 1) or Days 8 to 28 (1, 2.5,10 and 25 mg Compound A/placebo—Groups 2 to 5). All doses wereadministered at the study center after a standard breakfast. Subjects ineach dose group were allocated to study treatment in a ratio of 10Compound A to 2 placebo. Blood samples for pharmacokinetic andpharmacodynamic (CETP activity, CETP concentration, HDL-C, LDL-C, totalcholesterol, triglycerides) assessments were collected from prior toeach dose and at intervals throughout the study until 336 hours afterthe last dose. Secondary pharmacodynamic endpoints (includingapolipoproteins A1, A2, B, and E, HDL2-C, HDL3-C, phospholipids,HDL-free cholesterol [HDL-FC], HDL-cholesteryl ester [HDL-CE],HDL-phospholipids [HDL-PL], HDL-triglycerides [HDL-TG], and LDL particlesize) were measured at intervals until the last day of dosing. Urine wascollected for pharmacokinetics from pre dose and at intervals up to 72hours after the first and last dose. Safety assessments includingadverse events, blood pressure and pulse rate, ECGs, laboratory safetytests (including aldosterone) and physical examinations were conductedthroughout both studies.

Analytical Methods

Plasma and urine concentrations of Compound A were determined usingvalidated liquid chromatography with tandem mass spectrometry (LC/MS/MS)methods. The lower limit of quantification (LLQ) for both assays was0.500 ng/mL. Plasma concentration of CETP was determined using avalidated enzyme-linked immunosorbent assay (ELISA) method with a lowerlimit of quantification (LLQ) of 0.500 μg/mL. CETP activity wasdetermined as the rate of [³H]-labeled CE transfer from donor HDL toacceptor VLDL/LDL. [³H]CE-labeled HDL was added to human plasma andincubated for 4 hours at 37° C. Non-HDL lipoproteins were precipitatedand separated from HDL, and the amount of radioactivity in thesupernatant was quantitated. CETP activity was determined as thedifference of the radioactivity between the samples incubated at 37° C.and 4° C. HDL-C and LDL-C were measured by homogenous enzymaticcolorimetric assay using a Modular analyser (Roche Diagnostics). Totalcholesterol and triglycerides were measured by homogenous enzymaticassay using a Modular analyser (cholesterol oxidaseperoxidase-peroxidase aminophenazone phenol [CHOP-PAP]) method and aglycerol phosphate oxidase [GPO-PAP] method, respectively. ApoA1, ApoA2,ApoB and ApoE were measured by immunoturbidimetry using reagents fromRolf Greiner Biochemica (Germany) and N-apoprotein standard serum fromSiemens (Germany). LDL particle size was determined by gradient gelelectrophoresis. HDL fraction was separated by a combinedultracentrifugation-precipitation method (Beta-quantification). HDL-2and HDL-3 fractions were then separated by further ultracentrifugation.Total-cholesterol in HDL, HDL-2 and HLD-3 fractions, free cholesterol inHDL fraction, triglycerides in HDL fraction and phospholipids in plasmaand HDL-fraction were measured using enzymatic methods and reagents fromDiasys Diagnostics (Germany). The measurements were performed on anOlympus AU600 automatic analyzer and were calibrated using secondarystandards from Roche Diagnostics (Total-cholesterol, triglycerides) andDiasys Diagnostics (free cholesterol, phospholipids), respectively.Esterified cholesterol was calculated as the difference betweentotal-cholesterol and free cholesterol.

Statistical Analyses

The sample sizes for the study were chosen based on practicalconsiderations rather than statistical power. The numbers of subjects ineach group were considered to be adequate to assess the main objectivesof each study. Subjects were allocated to Compound A or placebo in eachgroup by means of a computer-generated randomization code.Pharmacokinetic parameters were determined by non-compartmental methodsusing WinNonlin software version 4.1 (Pharsight Corporation, USA). Alldata were listed and summarized by treatment group using descriptivestatistics. In the study, maximum percent changes from baseline at eachCompound A dose level were compared with pooled placebo using an ANOVAmodel. All statistical analyses were conducted using SAS version 6.12 orhigher (SAS Institute Inc. USA).

Pharmacokinetic Results

In the study, plasma concentrations appeared to increase approximatelyproportionally to dose following single doses from 1 to 25 mg, althoughnon-proportionality was observed at steady state: 7-fold, 9-fold and12-fold increases in C_(min,ss), AUC_(0-tau,ss) and C_(max,ss),respectively, for a 25-fold increase in dose. T_(max) was independent ofdose with median values of 4 to 6 hours post-dose. Variability wasmoderate following single and multiple dosing with CVs for C_(max),C_(min) and AUC parameters being ≤33%. Visual inspection of troughconcentrations suggests that Compound A approached steady state within 1to 2 weeks of daily dosing. The mean terminal half-life of Compound Afollowing the last dose was 121 to 151 hours and was independent ofdose. A similar half-life was observed between single and multiple doseof 5 to 25 mg of Compound A, respectively. Compound A accumulated withonce daily dosing in a dose-dependent manner, with an approximately6-fold increase at 1 mg through to a 2-fold increase at 25 mg.

Pharmacodynamics Results

Baseline pharmacodynamic parameters were well balanced across treatmentgroups the study. Compound A strongly inhibited CETP activity in adose-dependent manner following both single and repeated dosing. Nearcomplete CETP inhibition was observed following repeated doses of 2.5,5, 10 to 25 mg once daily Compound A (˜92 to 99%) (Table 1). This levelof inhibition was maintained throughout the repeated dosing period andthe maximum effect of each dose was achieved within 1 week of once dailydosing. The duration of inhibition after the last dose wasdose-dependent, with activity approaching baseline levels by 2 weeksfollowing the lowest dose (1 mg), but still being approximately 50%below baseline at 2 weeks following 10 and 25 mg dosing. Although CETPactivity decreased with Compound A dosing, the concentration of CETPincreased in a dose-dependent manner following both single and multipledosing. CETP concentration increased from baseline by 2.5- to 2.8-foldafter 3 weeks of dosing with 10 mg and 25 mg once daily Compound A. CETPconcentrations declined in parallel with plasma drug concentrations.Following the cessation of Compound A dosing, such that concentrationswere approaching baseline values within 2 weeks following 1 mg and 5 mgCompound A , whereas concentrations were still approximately 1.4-foldhigher than baseline at 2 weeks following 10 mg and 25 mg Compound A.The maximum percent changes in CETP activity and CETP concentrationswere statistically significantly different from placebo (p<0.0001) atall Compound A dose levels (1 to 25 mg).

HDL-C concentrations increased in a dose-dependent manner followingmultiple dosing. Once daily Compound A at doses of 2.5 to 25 mg led tomarked increases from baseline HDL-C of approximately 96% up to 140%.LDL-C concentrations decreased in a dose-dependent manner with maximumdecreases from baseline of approximately 40% to 53% following 2.5 to 25mg once daily Compound A. The maximum percent changes from baseline werestatistically significantly different from placebo (p<0.0001) followingonce daily Compound A doses of 5 to 25 mg for HDL-C and following 10 and25 mg for LDL C. HDL-C and LDL-C concentrations started to returntowards baseline following cessation of Compound A dosing consistentwith the loss of CETP inhibition. There were trends indicatingdose-related increases in Apo A-1, Apo E, HDL2-C and HDL3-C, anddecreases in Apo B concentrations. Variability was high for all of thesevariables; nevertheless the data suggest that maximum effects may havebeen achieved with doses of 5 to 10 mg once daily Compound A. There wasno dose-related trend in Apo A2 or phospholipids, but there weredose-related increases in HDL-FC, HDL-CE and HDL-PL and decreases inHDL-TG across the dose range 1 to 10 mg with no further changes noted at25 mg Compound A. There were no noteworthy changes in LDL particle size.In addition, there was no evidence of any relevant effect of food, age,gender or ethnicity on the pharmacodynamic variables.

Safety

Repeated doses up 25 mg once daily were well tolerated in all subjects.There were no serious adverse events and no subjects withdrew because ofadverse events. There were no clinically significant effects on bloodpressure or heart rate, ECG variables, physical examination orlaboratory safety tests. In particular, Compound A had no effect onserum electrolyte or aldosterone concentrations.

TABLE 1 Maximum percent change from baseline for primary pharmacodynamicvariables for Compound A following repeated oral doses in Caucasian malehealthy subjects Maximum % change from Placebo Placebo Compound A dose(once daily) baseline in: (Group 1) (Groups 2-5) 1 mg 2.5 mg 5 mg 10 mg25 mg Steady State Data N = 1 N = 8 N = 10 N = 10 N = 10 N = 10 N = 10CETP activity 21.3 −8.9 −66.5 −91.6 −90.9 −97.6 −99.4 (14.7) (6.4) (6.4)(7.1) (2.9) (1.1) CETP 34.9 19.5 128 144 215 249 280 concentration(18.4) (34.3) (22.3) (50.9) (49.6) (47.4) HDL-C −2.7 10.7 37.9 95.6 118140 136 concentration (20.5) (20.6) (30.4) (33.2) (36.4) (43.9) LDL-C25.0 −8.8 −29.6 −39.1 −41.7 −43.6 −53.2 concentration (20.8) (6.9)(14.3) (11.4) (14.6) (15.0) Total cholesterol 1.4 −8.1 −12.7 −7.9 −1.43.7 7.5 (18.1) (7.1) (14.2) (12.4) (18.2) (17.2) Triglycerides −28.8−46.1 −50.2 −45.8 −20.3 −22.2 −31.4 (20.9) (16.2) (16.5) (20.2) (19.9)(25.8) Values are mean (SD) Group 1 received 5 mg Compound A/placebo onDay 1 and Days 8 to 42. Groups 2-5 received 1, 2.5, 10 and 25 mgCompound A/placebo on Day 1 and Days 8 to 35. CHEMICAL NAME AND FORMULAOF COMPOUND A

{4-[(2-{[3,5-bis(trifluoromethyl)benzyl][(2R,4S)-1-(ethoxycarbonyl)-2-ethyl-6-(trifluoromethyl)-1,2,3,4-tetrahydroquinolin-4-yl]amino}pyrimidin-5-yl)oxy]butanoic acid}REFERENCES

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1-6. (canceled)
 7. A Pharmaceutical composition comprising atherapeutically effective amount of compound A:

or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient, wherein the composition is to beadministered orally to a subject in need thereof and wherein the dosageof said Compound A to be administered to said subject ranges from about1 mg to 25 mg per day.
 8. (canceled)
 9. The Pharmaceutical compositionfor use according to claim 7, wherein the dose of Compound A to beadministered to said subject in need thereof ranges from about 5 mg to10 mg per day.
 10. The Pharmaceutical composition according to any oneof claims 9, wherein the dose of Compound A to be administered to saidsubject in need thereof is about 5 mg per day or about 10 mg per day.11. The Pharmaceutical composition according to claim 9, wherein thecomposition is to be administered to said subject in need thereof for 1,5, 10, 20, 40, 52, 100, or 200 weeks.
 12. The Pharmaceutical compositionaccording to claim 9, wherein the composition is to be administered tosaid subject in need thereof for at least one week, or at least threeweeks.
 13. The Pharmaceutical composition according to claim 9, whereinthe pharmaceutical composition is formulated as a single unit dosageform.
 14. The Pharmaceutical composition according to claim 13, whereinthe single unit dosage form comprises about 1 mg to 25 mg of Compound A,or about 5 mg to 10 mg of Compound A, or about 5 mg or about 10 mg ofCompound A.
 15. The Pharmaceutical composition according to claim 13,wherein the composition is formulated as a solid oral dosage form. 16.The Pharmaceutical composition according to claim 13, wherein thecomposition is formulated as a tablet or capsule.
 17. The Pharmaceuticalcomposition according to claim 7, for use in the treatment of subjectssuffering from or having an increased risk for hyperlipidemia or mixeddyslipidemia. 18-20. (canceled)
 21. The Pharmaceutical compositionaccording to claim 13, wherein the composition is formulated as a liquidoral dosage form or as a solid oral dosage form, selected from a tabletor capsule.
 22. (canceled)